Arsenical Fluorescent Agents and Assays

ABSTRACT

The invention provides methods and compositions for labeling dithiol-containing analytes, including a substituted, optionally hydro-, optionally hetero-, monoarsenical anthracene compound comprising a 4′ rotation blocking group and a 5′ arsenic, and that exhibits a detectable increase or shift in fluorescence when the arsenic reacts with two thiols of a rotation-blocking binding target molecule.

This application is a continuation of Ser No. 13/053,206, filed: Mar.21, 2011 (now, U.S. Pat. No. 8,664,009), which claims priority to U.S.61/315,723 filed Mar. 19, 2010.

BACKGROUND OF THE INVENTION

The field of the invention is arsenical fluorescent agents anddiagnostic assays.

Neglected Tropical Diseases (NTDs) are a group of infections that mostcommonly plague those who are extremely poor and live in remote ruralareas, urban slums, and places of political conflict. Because theyadversely affect child development, pregnancy, and worker productivity,NTDs are a major reason why many in developing nations cannot escapeextreme poverty. Three such diseases are caused by trypanosomalparasites: Chagas' disease, African Trypanosomiasis or SleepingSickness, and Leishmaniasis.

While these diseases manifest in several clinical forms they are causedby a family of parasites with conserved biochemical machinery. We haveexploited these similarities to develop a new diagnostic to beadministered at the point of care (POC). In the case of these NTDs, thePOC can be anywhere from a remote village along the Amazon to a forwardoperating base in Iraq. Thus, POC diagnostics must perform well in lowtechnology settings with minimal requisite technical training. Sincerapid and accurate diagnosis is critical for any disease controlstrategy, new diagnostics for these diseases are needed for betteridentification and monitoring of treatment populations.

There are two classes of diagnostics for these diseases: (1)parasitological methods that detect the parasite directly; thesetypically require observation of the parasite in blood samples under amicroscope, and are labor intensive, time consuming, and are ofteninadequate for diagnosis in the chronic phase of infection when parasitelevels in the blood are lower; and (2) immunological methods that relyon detection of markers from a patient's immune response; these methodsare very sensitive but require highly trained technicians and expensivetechnology making them impractical for a field setting.

We have developed a new parasitological method that eliminates the needfor trained personnel and expensive instrumentation, instead using ahighly specific molecular sensor that can be detected with a handheld UVlamp or black light.

Immobilized monoarsenical [aminohexanoyl-4-aminophenylarsineoxide]supports have been described [Kalef et al., Anal Biochem. 1993 Aug. 1;212(2):325-34; Kalef et al., Methods Enzymol. 1994, 233, 395-403] forthe purification of proteins, and Adams et al., (J. Am. Chem. Soc.,2002, 124 (21), pp 6063-6076, 6068, col.2, line 5) describes afluorescein with just one arsenic substituent,4′-(1,3,2-dithiarsolan-2-yl)-5-carboxyfluorescein; see also, Hoffman etal., Nat. Protoc. 2010 September; 5(10):1666-77. Epub 2010 Sep. 23,“Fluorescent labeling of tetracysteine-tagged proteins in intactcells.”.

SUMMARY OF THE INVENTION

The invention provides methods and compositions for labelingdithiol-containing analytes. In one embodiment, the invention provides asubstituted, optionally hydro-, optionally hetero-, monoarsenicalanthracene compound comprising a 4′ rotation blocking group and a 5′arsenic, and that exhibits a detectable increase or shift influorescence when the arsenic reacts with two thiols of arotation-blocking binding target molecule, the compound having thestructure I:

wherein:

R1 is O, S, NRα, or CRα₂;

R2 is C or N;

R3 is optionally substituted-, optionally hetero-alkyl, optionallysubstituted-, optionally hetero-alkenyl, optionally substituted-,optionally hetero-alkynyl, optionally substituted-, optionallyhetero-aryl, or optionally substituted-, optionally hetero-alkoxy;

R4 and R5 are independently carbonyl, carbonothioyl, NRα₂, ORα, or SRα;

R6 and R7 are Rα, halide, ORα, NRα₂, ORα, SRα, or nitro (—NO₂);

R8 and R9 are Rα, halide, ORα, NRα₂, ORα, SRα, or nitro (—NO₂);

R10 is a rotational blocking group;

R11 is an arsenic protecting group displaced by reaction of the arsenicwith the thiols of the target molecule;

Rα groups are independently H, optionally substituted-, optionallyhetero-alkyl, optionally substituted-, optionally hetero-alkenyl,optionally substituted-, optionally hetero-alkynyl, optionallysubstituted-, optionally hetero-aryl, or optionally substituted-,optionally hetero-alkoxy;

wherein one or more of the pairs R4-R6, R6-R8, R5-R7, and R7-R9 may becovalently joined in one or more rings;

and tautomers, anhydrides and salts of the compound.

The invention encompasses all combinations of disclosed particularembodiments thereof, including wherein: R11 is carbonyl, 1,2ethanedithiol, or dihydroxyl, the rotation blocking group is anoptionally substituted-, optionally hetero-alkyl, optionallysubstituted-, optionally hetero-alkenyl, optionally substituted-,optionally hetero-alkynyl, optionally substituted-, optionallyhetero-aryl, or optionally substituted-, optionally hetero-alkoxy, andor the anthracene compound is a fluoresein, rhodamine, eosin, phenazine,phenoxazine, phenothiazine, thioxanthene, acridine, or a core orderivative thereof.

In a more general embodiment, the invention provides a substituted,optionally hydro-, optionally hetero-, monoarsenical anthracene compoundcomprising a 4′ rotation-blocking group and a 5′ arsenic, and thatexhibits a detectable increase or shift in fluorescence when the arsenicreacts with two thiols of a rotation-blocking binding target moleculeforming a conjugate having the general structure II:

wherein AC is the anthracene core, RB is the rotation blocking group andTM is the target molecule, and tautomers, anhydrides and salts of thecompound.

The invention also provide methods of making and using the subjectcompounds, including a method of using a subject compound comprising thestep of: contacting the compound with the target molecule wherein thearsenic reacts with the thiols, and in particular, a method ofdiagnosing a trypanasomatid infection in a patient by detecting adicysteine trypanathione, comprising the steps of: (a) contacting asample of the patient with a subject compound; and (b) detecting theincrease or shift in fluorescence as an indication of the presence ofthe trypanathione and the trypanasomatid infection.

The invention also provides a method of diagnosing a trypanasomatidinfection in a patient by detecting a dicysteine trypanathione,comprising the steps of: (a) contacting a sample of the patient with anarsenical fluorescent dye that exhibits a detectable increase or shiftin fluorescence when the arsenic of the dye reacts with the cysteines ofthe trypanathione; and (b) detecting the increase or shift influorescence as an indication of the presence of the trypanathione andthe trypanasomatid infection.

The subject monoarsenical dyes have a significant advantage overdiarsenical dyes in diagnoses, in that they form a brightly fluorescentcomplex upon binding of a dicysteine motif, whereas he 1:1 complex withprior diarsenical dyes is only weakly fluorescent. This in not a flaw ofthe diarsenicals, but reflect their distinct design criteria.Biarsenicals are used in excess to track a small concentration ofproteins as they are trafficked through cells, and hence requireexquisite selectivity and high binding affinity afforded by thetetradentate binding capacity of the bisarsenical. Essentially, thebiarsenticals were engineered as a small molecule equivalent of GFP tomonitor protein trafficking. In contrast, our diagnostic goals requireda probe much more sensitive, and the duration of binding event is not ascritical. Accurate diagnosis needs to detect a metabolite with twoproximal cysteines selectively, and employing a monoarsenical means thata 1:1 binding stoichiometry can provide a fluorescent conjugate which ismore favorable in this context. Furthermore, use of the blocking groupallows the binding event to provide a much brighter conjugate since freerotation about the arsenic carbon bond is more hindered and thereforefluorescence is less quenched.

In addition the invention provides all recombinations of alternativerecited elements as if each recombination were separately set forth.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The invention provides a substituted, optionally hydro-, optionallyhetero-, monoarsenical anthracene compound comprising a 4′rotation-blocking group and a 5′ arsenic, and that exhibits a detectableincrease or shift in fluorescence when the arsenic reacts with twothiols of a rotation-blocking binding target molecule forming aconjugate having the general structure II:

wherein AC is the anthracene core, RB is the rotation blocking group andTM is the target molecule, and tautomers, anhydrides and salts of thecompound.

In a more specific embodiment of Structure II, the invention provides asubstituted, optionally hydro-, optionally hetero-, monoarsenicalanthracene compound comprising a 4′ rotation blocking group and a 5′arsenic, and that exhibits a detectable increase or shift influorescence when the arsenic reacts with two thiols of arotation-blocking binding target molecule, the compound having thestructure I:

wherein:

R1 is O, S, NRα, or CRα₂;

R2 is C or N;

R3 is optionally substituted-, optionally hetero-alkyl, optionallysubstituted-, optionally hetero-alkenyl, optionally substituted-,optionally hetero-alkynyl, optionally substituted-, optionallyhetero-aryl, or optionally substituted-, optionally hetero-alkoxy;

R4 and R5 are independently carbonyl, carbonothioyl, NRα₂, ORα, or SRα;

R6 and R7 are Rα, halide, ORα, NRα₂, ORα, SRα, or nitro (—NO₂);

R8 and R9 are Rα, halide, ORα, NRα₂, ORα, SRα, or nitro (—NO₂);

R10 is a rotational blocking group;

R11 is an arsenic protecting group displaced by reaction of the arsenicwith the thiols of the target molecule;

Rα groups are independently H, optionally substituted-, optionallyhetero-alkyl, optionally substituted-, optionally hetero-alkenyl,optionally substituted-, optionally hetero-alkynyl, optionallysubstituted-, optionally hetero-aryl, or optionally substituted-,optionally hetero-alkoxy;

wherein one or more of the pairs R4-R6, R6-R8, R5-R7, and R7-R9 may becovalently joined in one or more rings;

and tautomers, anhydrides and salts of the compound.

The following descriptions of particular embodiments and examples areprovided by way of illustration and not by way of limitation. Thoseskilled in the art will readily recognize a variety of noncriticalparameters that could be changed or modified to yield essentiallysimilar results. Unless contraindicated or noted otherwise, in thesedescriptions and throughout this specification, the terms “a” and “an”mean one or more, the term “or” means and/or and polynucleotidesequences are understood to encompass opposite strands as well asalternative backbones described herein. Furthermore, genuses are recitedas shorthand for a recitation of all members of the genus; for example,the recitation of (C1-C3) alkyl is shorthand for a recitation of allC1-C3 alkyls: methyl, ethyl and propyl, including isomers thereof.

The term “heteroatom” as used herein generally means any atom other thancarbon, hydrogen or oxygen. Preferred heteroatoms include oxygen (O),phosphorus (P), sulfur (S), nitrogen (N), silicon (S), arsenic (As),selenium (Se), and halogens, and preferred heteroatom functional groupsare haloformyl, hydroxyl, aldehyde, amine, azo, carboxyl, cyanyl,thocyanyl, carbonyl, halo, hydroperoxyl, imine, aldimine, isocyanide,iscyante, nitrate, nitrile, nitrite, nitro, nitroso, phosphate,phosphono, sulfide, sulfonyl, sulfo, and sulfhydryl.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which is fully saturated,having the number of carbon atoms designated (i.e. C1-C8 means one toeight carbons). Examples of alkyl groups include methyl, ethyl,n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl and the like.

The term “alkenyl”, by itself or as part of another substituent, means astraight or branched chain, or cyclic hydrocarbon radical, orcombination thereof, which may be mono- or polyunsaturated, having thenumber of carbon atoms designated (i.e. C2-C8 means two to eightcarbons) and one or more double bonds. Examples of alkenyl groupsinclude vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl) and higher homologs and isomersthereof.

The term “alkynyl”, by itself or as part of another substituent, means astraight or branched chain hydrocarbon radical, or combination thereof,which may be mono- or polyunsaturated, having the number of carbon atomsdesignated (i.e. C2-C8 means two to eight carbons) and one or moretriple bonds. Examples of alkynyl groups include ethynyl, 1- and3-propynyl, 3-butynyl and higher homologs and isomers thereof.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from alkyl, as exemplified by—CH₂—CH₂—CH₂—CH₂—. Typically, an alkyl (or alkylene) group will havefrom 1 to 24 carbon atoms, with those groups having 10 or fewer carbonatoms being preferred in the invention. A “lower alkyl” or “loweralkylene” is a shorter chain alkyl or alkylene group, generally havingeight or fewer carbon atoms.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and from one to three heteroatoms selectedfrom the group consisting of O, N, Si and S, wherein the nitrogen andsulfur atoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) O, N and S may be placed atany interior position of the heteroalkyl group. The heteroatom Si may beplaced at any position of the heteroalkyl group, including the positionat which the alkyl group is attached to the remainder of the molecule.Examples include —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃,—CH₂—S—CH₂—CH₃, —CH₂—CH₂, —S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃,—Si(CH₃)₃, —CH₂—CH═N—OCH₃, and —CH═CH—N(CH3)—CH₃. Up to two heteroatomsmay be consecutive, such as, for example, —CH₂—NH—OCH₃ and—CH₂—O—Si(CH₃)₃.

Similarly, the term “heteroalkylene,” by itself or as part of anothersubstituent means a divalent radical derived from heteroalkyl, asexemplified by —CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. Forheteroalkylene groups, heteroatoms can also occupy either or both of thechain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino,alkylenediamino, and the like). Still further, for alkylene andheteroalkylene linking groups, no orientation of the linking group isimplied.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Accordingly, acycloalkyl group has the number of carbon atoms designated (i.e., C3-C8means three to eight carbons) and may also have one or two double bonds.A heterocycloalkyl group consists of the number of carbon atomsdesignated and from one to three heteroatoms selected from the groupconsisting of O, N, Si and S, and wherein the nitrogen and sulfur atomsmay optionally be oxidized and the nitrogen heteroatom may optionally bequaternized. Additionally, for heterocycloalkyl, a heteroatom can occupythe position at which the heterocycle is attached to the remainder ofthe molecule. Examples of cycloalkyl include cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include 1-(1,2,5,6-tetrahydropyrid-yl), 1-piperidinyl,2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl,tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl,tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like.

The terms “halo” and “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include alkyl substituted with halogen atoms, which can be thesame or different, in a number ranging from one to (2m′+1), where m′ isthe total number of carbon atoms in the alkyl group. For example, theterm “halo(C1-C4)alkyl” is mean to include trifluoromethyl,2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like. Thus,the term “haloalkyl” includes monohaloalkyl (alkyl substituted with onehalogen atom) and polyhaloalkyl (alkyl substituted with halogen atoms ina number ranging from two to (2m′+1) halogen atoms, where m′ is thetotal number of carbon atoms in the alkyl group). The term“perhaloalkyl” means, unless otherwise stated, alkyl substituted with(2m′+1) halogen atoms, where m′ is the total number of carbon atoms inthe alkyl group. For example the term “perhalo(C1-C4)alkyl” is meant toinclude trifluoromethyl, pentachloroethyl,1,1,1-trifluoro-2-bromo-2-chloroethyl and the like.

The term “acyl” refers to those groups derived from an organic acid byremoval of the hydroxy portion of the acid. Accordingly, acyl is meantto include, for example, acetyl, propionyl, butyryl, decanoyl, pivaloyl,benzoyl and the like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,typically aromatic, hydrocarbon substituent which can be a single ringor multiple rings (up to three rings) which are fused together or linkedcovalently. Non-limiting examples of aryl groups include phenyl,1-naphthyl, 2-naphthyl, 4-biphenyl and 1,2,3,4-tetrahydronaphthalene.

The term heteroaryl,” refers to aryl groups (or rings) that contain fromzero to four heteroatoms selected from N, O, and S, wherein the nitrogenand sulfur atoms are optionally oxidized and the nitrogen heteroatom areoptionally quaternized. A heteroaryl group can be attached to theremainder of the molecule through a heteroatom. Non-limiting examples ofheteroaryl groups include 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl,4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyland 6-quinolyl.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) is meant to include both substituted and unsubstitutedforms of the indicated radical. Preferred substituents for each type ofradical are provided below.

Substituents for the alkyl and heteroalkyl radicals (as well as thosegroups referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl,alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl andheterocycloalkenyl) can be a variety of groups selected from: —OR′, ═O,═NR′, ═N—OR′, —NR′R″, —SR′, halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′,—CO₂R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″,—NR′—SO₂NR′″, —NR″CO₂R′, —NH—C(NH₂)═NH, —NR′C(NH₂)═NH, —NH—C(NH₂)═NR′,—S(O)R′, —SO₂R′, —SO₂NR′R″, —NR″SO₂R, —CN and —NO₂, in a number rangingfrom zero to three, with those groups having zero, one or twosubstituents being particularly preferred. R′, R″ and R′″ eachindependently refer to hydrogen, unsubstituted (C1-C8)alkyl andheteroalkyl, unsubstituted aryl, aryl substituted with one to threehalogens, unsubstituted alkyl, alkoxy or thioalkoxy groups, oraryl-(C1-C4)alkyl groups. When R′ and R″ are attached to the samenitrogen atom, they can be combined with the nitrogen atom to form a 5-,6- or 7-membered ring. For example, —NR′R″ is meant to include1-pyrrolidinyl and 4-morpholinyl. Typically, an alkyl or heteroalkylgroup will have from zero to three substituents, with those groupshaving two or fewer substituents being preferred in the invention. Morepreferably, an alkyl or heteroalkyl radical will be unsubstituted ormonosubstituted. Most preferably, an alkyl or heteroalkyl radical willbe unsubstituted. From the above discussion of substituents, one ofskill in the art will understand that the term “alkyl” is meant toinclude groups such as trihaloalkyl (e.g., —CF₃ and —CH₂CF₃).

Preferred substituents for the alkyl and heteroalkyl radicals areselected from: —OR′, ═O, —NR′R″, —SR′, halogen, —SiR′R″R′″, —OC(O)R′,—C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR″CO₂R′,—NR′—SO₂NR″R′″, —S(O)R′, —SO2R′, —SO₂NR′R″, —NR″SO₂R, —CN and —NO₂,where R′ and R″ are as defined above. Further preferred substituents areselected from: —OR′, ═O, —NR′R″, halogen, —OC(O)R′, —CO₂R′, —CONR′R″,—OC(O)NR′R″, —NR″C(O)R′, —NR″CO₂R′, —NR′—SO₂NR″R′″, —SO₂R′, —SO₂NR′R″,—NR″SO₂R, —CN and —NO₂.

Similarly, substituents for the aryl and heteroaryl groups are variedand selected from: halogen, —OR′, —OC(O)R′, —NR′R″, —SR′, —R′, —CN,—NO₂, —CO₂R′, —CONR′R″, —C(O)R′, —OC(O)NR′R″, —NR″C(O)R′, —NR″CO2R′,—NR′—C(O)NR″R′″, —NR′—SO₂NR″R′″, —NH—C(NH2)═NH, —NR′C(NH₂)═NH,—NH—C(NH₂)═NR′, —S(O)R′, —SO₂R′, —SO₂NR′R″, —NR″SO₂R, —N₃, —CH(Ph)₂,perfluoro(C1-C4)alko-xy and perfluoro(C1-C4)alkyl, in a number rangingfrom zero to the total number of open valences on the aromatic ringsystem; and where R′, R″ and R′″ are independently selected fromhydrogen, (C1-C8)alkyl and heteroalkyl, unsubstituted aryl andheteroaryl, (unsubstituted aryl)-(C1-C4)alkyl and (unsubstitutedaryl)oxy-(C1-C4)alkyl. When the aryl group is1,2,3,4-tetrahydronaphthalene, it may be substituted with a substitutedor unsubstituted (C3-C7)spirocycloalkyl group. The(C3-C7)spirocycloalkyl group may be substituted in the same manner asdefined herein for “cycloalkyl”. Typically, an aryl or heteroaryl groupwill have from zero to three substituents, with those groups having twoor fewer substituents being preferred in the invention. In oneembodiment of the invention, an aryl or heteroaryl group will beunsubstituted or monosubstituted. In another embodiment, an aryl orheteroaryl group will be unsubstituted.

Preferred substituents for aryl and heteroaryl groups are selected from:halogen, —OR′, —OC(O)R′, —NR′R″, —SR′, —R′, —CN, —NO₂, —CO₂R′, —CONR′R″,—C(O)R′, —OC(O)NR′R″, —NR″C(O)R′, —S(O)R′, —SO₂R′, —SO₂NR′R″, —NR″SO₂R,—N₃, —CH(Ph)₂, perfluoro(C1-C4)alkoxy and perfluoro(C1-C4)alkyl, whereR′ and R″ are as defined above. Further preferred substituents areselected from: halogen, —OR′, —OC(O)R′, —NR′R″, —R′, —CN, —NO₂, —CO₂R′,—CONR′R″, —NR″C(O)R′, —SO₂R′, —SO₂NR′R″, —NR″SO₂R,perfluoro(C1-C4)alkoxy and perfluoro(C1-C4)alkyl.

The substituent —CO₂H, as used herein, includes bioisostericreplacements therefor; see, e.g., The Practice of Medicinal Chemistry;Wermuth, C. G., Ed.; Academic Press: New York, 1996; p. 203.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CH₂)_(q)—U—, wherein T and U are independently —NH—, —O—, —CH₂—or a single bond, and q is an integer of from 0 to 2. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula -A-(CH2)r-B—,wherein A and B are independently —CH₂—, —O—, —NH—, —S—, —S(O)—,—S(O)₂—, —S(O)₂NR′— or a single bond, and r is an integer of from 1 to3. One of the single bonds of the new ring so formed may optionally bereplaced with a double bond. Alternatively, two of the substituents onadjacent atoms of the aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula —(CH₂)s-X—(CH₂)t-, where s and t areindependently integers of from 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—,—S(O)₂—, or —S(O)₂NR′—. The substituent R′ in —NR′— and —S(O)₂NR′— isselected from hydrogen or unsubstituted (C1-C6)alkyl.

In both embodiments, the rotation blocking group (RBC or R10) is bulkygroup that limits rotation of the substituent at the 4′ carbon ofanthracene ring core sufficient to substantially reduce fluorescentquenching. A proton (H), F, or other small, or small, linearsubstituents are insufficiently bulky to quench fluorescence, as is the-AsEDT group of the biarsenicals discussed below. Preferred blockinggroups are large, bulky, tetrahedral, and/or aryl functional groups,particularly optionally substituted-, optionally hetero-alkyl,optionally substituted-, optionally hetero-alkenyl, optionallysubstituted-, optionally hetero-alkynyl, optionally substituted-,optionally hetero-aryl, or optionally substituted-, optionallyhetero-alkoxy.

What is important is that the blocking group quench the fluorescence ofthe compound sufficiently so that when the arcenic atom binds the thiolsof the binding target, a practically detectable increase or shift influorescence results. Without being bound by the theory, the idea isthat when the metabolite binds, there is more bulk about the arseniccarbon bond. If there is a substituent in the 4′/5′ position, the newlyformed ring system will be very bulky and its free rotation will beprevented by the presence of that blocking group. When you limit freerotation, you decrease the efficiency of the quenching process,effectively unquenching the fluorescnce. In practice syntheticaccessibility, convenience, resultant chemical and fluorometricproperties, and compatibility with the intended binding target assayeffectively guide the selection the blocking group. Accordingly, thetarget molecule, when bound to the compound though a pair of thiols,such as a dicysteine, similarly inhibits rotation at the 5′ carbon (notethat the 4′ and 5′ positions can be reversed, depending on the tautomer)of anthracene ring core sufficient to substantially reduce fluorescentquenching.

The 5′ arsenic atom is typically protected with an arsenic protectinggroup (R11) displaced by reaction of the arsenic with the thiols of thetarget molecule. This protecting group should be fluorescent quenching(should not limit free rotation), and is hence, preferably a relativelysmall, non-bulky group like carbonyl or dihydroxyl, and particulary,small dithiols like 1,2 ethanedithiol. What is important is that targetbinding provides a detectable increase or shift in fluorescence comparedwith the protecting group (e.g. EDT)-bound As. The protecting groups maybe used to protect the arsenical molecule from reacting with lowaffinity sites. Dithiol protecting groups may form a five- orsix-membered ring with the arsenic. Vicinal dithiols that form rings,such as 5-membered rings, are preferable. Dithiols that contain ringsmay increase the affinity of the dithiol to the arsenic by organizingthe two thiol groups to be in a cis-conformation ready to form anadditional ring with the arsenic. Examples of dithiol rings are 1,2benzenedithiol and 1,2-cyclohexanedithiol. Preferably, the arsenic isbonded to a dithiol, such as 1,2-ethanedithiol (EDT).

The subject compounds encompass the core structures of a wide variety ofanthracene-based fluorescent molecules including fluorescein, rhodamine,eosin, phenazine, phenoxazine, phenothiazine, thioxanthene, acridine,and cores and derivatives thereof. A wide variety of such anthracenecores are commercially available or readily synthesized by those skilledin the art. For example, Sigma-Aldrich lists dozens of fluoresceins andderivatives:

Product# Description 072171-(O′-Methylfluoresceinyl)piperidine-4-carboxylic acid 358452′,7′-Dichlorofluorescein diacetate C7153 5(6)-Carboxyfluorescein Mixedisomers C8166 5(6)-Carboxyfluorescein diacetate Mixed isomers 218885(6)-Carboxyfluorescein diacetate N-succinimidyl ester D05315-([4,6-Dichlorotriazin-2-yl]amino)fluorescein hydrochloride 727555-(Bromomethyl)fluorescein I9271 5-(Iodoacetamido)fluorescein 874445-Carboxy-fluorescein diacetate N-succinimidyl ester 868265-Carboxyfluorescein 92846 5-Carboxyfluorescein N-succinimidyl esterD9908 6-([4,6-Dichlorotriazin-2-yl]amino)fluorescein hydrochloride C41096-Carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein N-hydroxysuccinimideester 08951 6-Carboxy-fluorescein diacetate N-succinimidyl ester C06626-Carboxyfluorescein 54115 6-Carboxyfluorescein 469356-[Fluorescein-5(6)-carboxamido]hexanoic acid 469406-[Fluorescein-5(6)-carboxamido]hexanoic acid N-hydroxysuccinimide esterE6003 Eosin Y disodium salt E4382 Eosin Y disodium salt 32617 Eosin Ydisodium salt 45240 Eosin Y disodium salt 318906 Eosin Y solution F2456Fluorescein (free acid) F3651 Fluorescein 5(6)-isothiocyanate 46950Fluorescein 5(6)-isothiocyanate 46939 Fluorescein diacetate 5-maleimide36438 Fluorescein diacetate 6-isothiocyanate F7250 Fluoresceinisothiocyanate isomer I F4274 Fluorescein isothiocyanate isomer I 46951Fluorescein isothiocyanate isomer I F2502 Fluorescein isothiocyanateisomer I 46980 Fluorescein mercuric acetate 28803 Fluorescein sodiumsalt 46970 Fluorescein sodium salt F9551 Fluorescein-5-EXN-hydroxysuccinimide ester 88596 Fluorescein-O′-acetic acid 07294O′-(Carboxymethyl)fluoresceinamide

Generating other suitable anthracene derivatives is a mature art, withwell-established protocols, e.g. Hermanson, 2008, Bioconjugatetechniques (Academic Press, 2008); Afonso et al., Chem. Soc. Rev., 2009,38, 2410-2433 “Synthesis and applications of Rhodamine derivatives asfluorescent probes”, Jose et al. Tetrahedron 62 (2006) 11021-11037,“Benzophenoxazine-based fluorescent dyes for labeling biomolecules”.

The subject compounds encompass monoarsenical forms of the biarsenicalcompounds disclosed in U.S. Pat. No. 7,776,999, U.S. Pat. No. 7,524,972,U.S. Pat. No. 7,138,503, U.S. Pat. No. 6,900,304, U.S. Pat. No.6,686,458, U.S. Pat. No. 6,451,569, U.S. Pat. No. 6,054,271, U.S. Pat.No. 6,008,378, U.S. Pat. No. 5,932,474, wherein the 4′ arsenical groupof the biarsenical is replaced with a 4′ rotation blocking group asdisclosed herein, and the compound provides the request fluorescence andbinding.

For subject diagnostic assay use, the anthracene is initially selectedfor its optical properties, and may be optimized according to thefollowing criteria: (1) ability to form a fluorescent conjugate with asingle molecule of TSH2; (2) brightness of the monoarsenical-TSH2conjugate; (3) wavelength of absorption (λabs) and the wavelength ofemission (λems); (4) resistance to photobleaching; and (5) rate ofconjugation with TSH2. Any monoarsenical dye that has λ0.5% of thefluorescence of the monoarsenical-TSH2 conjugate, forms a bright complexin a short time period, and has a maximum λabs in the UVA range (320-400nm, the optical range of a commercially available black light) should beconfirmed for use and compatibility in serum extracts.

In particular embodiments the compounds encompass:

fluoresein-based compounds phenothiazine-based compounds wherein:wherein: R1 is O; R1 is S; R2 is C; R2 is N; R3 is 2-carboxyphenyl; R3is H or C; R4 is hydroxyl; R4 is hydroxyl; R5 is carbonyl; R5 iscarbonyl; R6 and R7 are H; and R6 and R7 are H; and R8 and R9 are H. R8and R9 are H fluoresein-based compounds thioxanthene-based compoundswherein: wherein: R1 is O; R1 is S; R2 is C; R2 is C; R3 is2-carboxyphenyl; R3 is 2-carboxyphenyl; R4 is hydroxyl; R4 is hydroxyl;R5 is carbonyl; R5 is carbonyl; R6 and R7 are H or Cl; R6 and R7 are H;and R8 and R9 are H; and R8 and R9 are H. R10 is methyl or benzylrhodamine-based compounds anthracene-based compounds wherein: wherein:R1 is O; R1 and R2 are C; R2 is C; R3 is H or C; R3 is 2-carboxyphenyl;R4 and R5 are O, S, N; R4 is amine; R6 and R7 are H; and R5 is amine; R8and R9 are H. R6 and R7 are H; and R8 and R9 are H eosin-based compoundsacridine-based compounds wherein: wherein: R1 is O; R1 is N; R2 is C; R2is C; R3 is 2-carboxyphenyl; R3 is H or C; R4 is hydroxyl; R4 and R5 areO, S, or N; R5 is carbonyl; R6 and R7 are H; and R6 and R7 are Br ornitro R8 and R9 are H. (—NO₂); and R8 and R9 are H. phenazine-basedcompounds acridine yellow (2,7-Dimethylacridine-3,6- wherein:diamine)-based compounds wherein: R1 and R2 are N; R1 is N; R3 is H ormethyl; R2 is C; R4 and R5 are O, S, or N; R3 is H; R6 and R7 are H; andR4 and R5 are NH₂; R8 and R9 are H R6 and R7 are CH₃; and R8 and R9 areH acridine orange (N,N,N′,N′- phenoxazine-basedTetramethylacridine-3,6-diamine)- compounds wherein: based compoundswherein: R1 is O; R1 is N; R2 is N; R2 is C; R3 is H or methyl; R3 is H;R4 is hydroxyl; R4 and R5 are N(CH₃)₂; R5 is carbonyl; R6 and R7 are H;and R6 and R7 are H; and R8 and R9 are H. R8 and R9 are H.

An exemplary synthetic scheme and exemplary compounds 1-4 are shownbelow.

The invention encompasses tautomers, anhydrides and salts of the subjectcompounds. For example, a generic 4′-5′ tautomer is shown here:

Furthermore, the structures below show two tautomers of compound 5 aswell as its salt form. In addition, one can hydrolyze to get the hydrateor dehydrate to get the arsenic oxide Compound 8 is the biotinconjugate, which may be used for example, in the construction of solidsupported dyes for use in the diagnostic.

The invention also encompasses method of making the subject compoundsand conjugates, and methods of using the subject compounds comprisingthe step of: (a) contacting the compound with the target moleculewherein the arsenic reacts with the thiols, and particularly. adicysteine. In a more particular embodiment, the invention provides amethod of diagnosing a trypanasomatid infection in a patient bydetecting a dicysteine trypanathione, comprising the steps of: (a)contacting a sample of the patient with a subject compound; and (b)detecting the increase or shift, particularly a red-shift, influorescence as an indication of the presence of the trypanathione andthe trypanasomatid infection.

In a separate embodiment, the invention provides a method of diagnosinga trypanasomatid infection in a patient by detecting a dicysteinetrypanathione, comprising the steps of: (a) contacting a sample of thepatient with an arsenical fluorescent dye that exhibits a detectableincrease or shift in fluorescence when the arsenic of the dye reactswith the cysteines of the trypanathione; and (b) detecting the increaseor shift in fluorescence as an indication of the presence of thetrypanathione and the trypanasomatid infection. This embodiment providesand encompasses a novel and unprecedented use of prior art biarsenicals.

EXAMPLES

Trypanosomal parasites regulate the oxidative environment inside theircells using a unique redox pair, the metabolite trypanothione (TSH₂),and the enzyme trypanothione reductase (TyR). TSH₂ was first describedas the potential target for arsenical therapeutics used to treat theseinfections. This pathway is orthogonal to that of glutathione andglutathione reductase (GR) found in the human patient or other mammals.Since the arsenical therapeutics have such a high affinity for thesulfur atoms in TSH₂, we hypothesized that arsenical dyes could besensitive and selective sensors for the detection of trypanosomalparasites.

To test this hypothesis, we employed the fluorescein arsenical helixbinder (FlAsH-EDT₂) dye first developed by Roger Tsien and coworkers forthe in vivo imaging of peptides and proteins possessing four cysteineamino acids. When unbound, FlAsH-EDT₂ is virtually non-fluorescent. Uponbinding of four sulfur atoms, free rotation about the As—C bond isrestricted, and the dye becomes brightly fluorescent. Since thisvisualization method labels proteins inside of cells with low backgroundfluorescence, we hypothesized that this molecular sensor could detectthe parasite metabolite without interference from other thiols presentin biological fluids. The binding of two molecules of TSH₂ shouldprevent the free rotation about the As—C bond in FlAsH, causing afluorimetric response.

FlAsH-EDT₂ was virtually non-fluorescent, but upon introduction of TSH₂,the fluorescence intensity rose. After approximately 10 minutes, thereaction reached saturation. Using the normalized fluorescencemeasurements, we found that FlAsH-EDT₂ had 0.4% of the fluorescence ofthe FlAsH-(TSH₂)₂ conjugate. The excitation and emission maxima for theconjugate were 505 nm and 527 nm respectively. This increase influorescence is readily detectable without a fluorimeter using a simplehandheld black light such as those used at airport security checkpoints.This detection method could be implemented easily in a low technology,resource poor setting. We examined the limits of detection of themetabolite with FlAsH-EDT₂. In our liquid-liquid method, a 10-foldexcess of the metabolite is sufficient to observe a fluorimetricresponse within twenty minutes. Therefore, 2.5 nmol of the dye can beused to detect 25 nmol of the metabolite. While lower concentrationswere detectable using a fluorimeter, the conjugation was too slow to bepractical. However, FlAsH-EDT₂ was not designed and is not optimized forthis purpose, and it requires two molecules of TSH₂ to bind in order toachieve the desired fluorescence response.

Hence we developed next generation dyes that have better opticalproperties and faster development times upon binding TSH₂. We focusedour efforts on the synthesis of a monoarsenical probe for the detectionof TSH₂. We hypothesized that if we could replace one of the sensordomains with a bulky group, binding of TSH₂ to the single sensor domainwould restrict free rotation and cause a fluorimetric response. Thishypothesis was verified. We synthesized a panel of dyes, which possessesa single arsenic nucleus and has a blocking group (such as a methylgroup) to prevent free rotation about the As—C bond. Indeed, conjugationof TSH₂ provides a measurable on response. There are a number offeatures that make our monoarsenicals better for a trypanosomaldiagnostic than FlAsH-EDT₂; for example, our dyes only requires a singlemetabolite to bind in order to obtain the desired fluorimetric responsedecreasing both the amount of dye required to detect the metabolite andthe time required for detection. This dramatic improvement translates toa better POC device.

To optimize the optical properties of our target dye, we initiallysynthesized a library of monoarsenical dyes from fluorescein andrhodamine scaffolds. Fluorescein, a well-studied imaging agent that hasbeen approved by the FDA, has strong fluorescence at low pH values andis the basis for FlAsH-EDT₂ and SRI-010346. Rhodamine dyes have morefavorable properties for our diagnostic purposes. Compared withfluorescein, they are relatively resistant to photobleaching and arefluorescent over a broad pH range of 4-10. Our data indicate the broadapplicability of our monoarsenicals to trypanosome diagnosis.

Scheme 2: Synthetic approach to ew monoarsenical dyes with differentoptical properties.

The invention encompasses all recombinations of alternative elements orcomponents as if each recombination were individually and belaboredlyset forth herein. The foregoing examples and detailed description areoffered by way of illustration and not by way of limitation. Allpublications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. Although the foregoing invention has beendescribed in some detail by way of illustration and example for purposesof clarity of understanding, it will be readily apparent to those ofordinary skill in the art in light of the teachings of this inventionthat certain changes and modifications may be made thereto withoutdeparting from the spirit or scope of the appended claims

What is claimed is:
 1. A substituted, optionally hydro-, optionallyhetero-, monoarsenical anthracene compound comprising a 4′ rotationblocking group and a 5′ arsenic, and that exhibits a detectable increaseor shift in fluorescence when the arsenic reacts with two thiols of arotation-blocking binding target molecule, the compound having thestructure I:

wherein: R1 is O, S, NRα, or CRα₂; R2 is C or N; R3 is H, optionallysubstituted-, optionally hetero-alkyl, optionally substituted-,optionally hetero-alkenyl, optionally substituted-, optionallyhetero-alkynyl, optionally substituted-, optionally hetero-aryl, oroptionally substituted-, optionally hetero-alkoxy; R4 and R5 areindependently carbonyl, carbonothioyl, NRα₂, ORα, or SRα; R6 and R7 areRα, halide, ORα, NRα₂, ORα, SRα, or nitro (—NO₂); R8 and R9 are Rα,halide, ORα, NRα₂, ORα, SRα, or nitro (—NO₂); R10 is a rotationalblocking group; R11 is an arsenic protecting group displaced by reactionof the arsenic with the thiols of the target molecule; Rα groups areindependently H, optionally substituted-, optionally hetero-alkyl,optionally substituted-, optionally hetero-alkenyl, optionallysubstituted-, optionally hetero-alkynyl, optionally substituted-,optionally hetero-aryl, or optionally substituted-, optionallyhetero-alkoxy; wherein one or more of the pairs R4-R6, R6-R8, R5-R7, andR7-R9 may be covalently joined in one or more rings; and tautomers,anhydrides and salts of the compound.
 2. The compound of claim 1 whereinR11 is carbonyl, 1,2 ethanedithiol, or dihydroxyl.
 3. The compound ofclaim 1 wherein the rotation blocking group is an optionallysubstituted-, optionally hetero-alkyl, optionally substituted-,optionally hetero-alkenyl, optionally substituted-, optionallyhetero-alkynyl, optionally substituted-, optionally hetero-aryl, oroptionally substituted-, optionally hetero-alkoxy.
 4. The compound ofclaim 1 wherein: R11 is carbonyl, 1,2 ethanedithiol, or dihydroxyl; andthe rotation blocking group is an optionally substituted-, optionallyhetero-alkyl, optionally substituted-, optionally hetero-alkenyl,optionally substituted-, optionally hetero-alkynyl, optionallysubstituted-, optionally hetero-aryl, or optionally substituted-,optionally hetero-alkoxy.
 5. The compound of claim 1 wherein: R1 is O,S, N, or C;
 6. The compound of claim 1 wherein: R1 is O, S, N, or C; R11is carbonyl, 1,2 ethanedithiol, or dihydroxyl; and the rotation blockinggroup is an optionally substituted-, optionally hetero-alkyl, optionallysubstituted-, optionally hetero-alkenyl, optionally substituted-,optionally hetero-alkynyl, optionally substituted-, optionallyhetero-aryl, or optionally substituted-, optionally hetero-alkoxy. 7.The compound of claim 1 wherein: R1 is O; R1 is O; R1 is O; R2 is C; R2is C; R2 is C; R3 is 2-carboxyphenyl; R3 is 2-carboxyphenyl; R3 is2-carboxyphenyl; R4 is hydroxyl; R4 is hydroxyl; R4 is amine; R5 iscarbonyl; R5 is carbonyl; R5 is amine; R6 and R7 are H; and R6 and R7are H or Cl; R6 and R7 are H; and R8 and R9 are H; R8 and R9 are H; andR8 and R9 are H; R10 is methyl or benzyl; R1 is O; R1 and R2 are N; R1is O; R2 is C; R3 is H or methyl; R2 is N; R3 is 2-carboxyphenyl; R4 andR5 are O, S, or N; R3 is H or methyl; R4 is hydroxyl; R6 and R7 are H;and R4 is hydroxyl; R5 is carbonyl; R8 and R9 are H; R5 is carbonyl; R6and R7 are Br or nitro (—NO₂); R6 and R7 are H; and and R8 and R9 are H;R8 and R9 are H; R1 is S; R1 is S; R1 and R2 are C; R2 is N; R2 is C; R3is H or C; R3 is H or C; R3 is 2-carboxyphenyl; R4 and R5 are O, S, N;R4 is hydroxyl; R4 is hydroxyl; R6 and R7 are H; and R5 is carbonyl; R5is carbonyl; R8 and R9 are H; R6 and R7 are H; and R6 and R7 are H; andR8 and R9 are H; R8 and R9 are H; R1 is N; R1 is N; R1 is N; R2 is C; R2is C; R2 is C; R3 is H or C; R3 is H; R3 is H; R4 and R5 are O, S, or N;R4 and R5 are NH₂; R4 and R5 are N(CH₃)₂; R6 and R7 are H; and R6 and R7are CH₃; and R6 and R7 are H; and R8 and R9 are H; R8 and R9 are H; orR8 and R9 are H.


8. The compound of claim 7 wherein: R11 is carbonyl, 1,2 ethanedithiol,or dihydroxyl; and the rotation blocking group is an optionallysubstituted-, optionally hetero-alkyl, optionally substituted-,optionally hetero-alkenyl, optionally substituted-, optionallyhetero-alkynyl, optionally substituted-, optionally hetero-aryl, oroptionally substituted-, optionally hetero-alkoxy.
 9. A compound ofclaim 1 selected from:


10. The compound of claim 1 wherein the arsenic is reacted with twothiols of a rotation-blocking binding target molecule forming aconjugate having the general structure II:

wherein AC is the anthracene core, RBG is the rotation blocking groupand TM is the target molecule, and tautomers, anhydrides and salts ofthe compound.
 11. A method of using a compound of claim 1 comprising thestep of: contacting the compound with the target molecule wherein thearsenic reacts with the thiols.